| Group | Attendee |
|---|---|
| Group 1 | Thushan de Silva |
| Saikou Y Bah | |
| Alex Keeley | |
| Group 2 | Joby Cole |
| Daniel Bose | |
| Frances Pick | |
| Group 3 | Ellie Harrison |
| Haya Almutairi | |
| Luke Geen | |
| Group 4 | Emmanuel Amabebe |
| Megan Cavanagh | |
| Neha Kulkarni |
We have a packed day of practical and bioinformatic analysis. Here is the rough plan:
| Start | End | Session | Handbook Page |
|---|---|---|---|
| 09:00 | 09:15 | Health & Safety Introducton and Risk Assessment | Page 2,12-18 |
| 09:15 | 09:45 | Introduction | |
| 09:45 | 10:30 | Quantification & Preparation of PCR Reaction | Pages 5-6 |
| 10:30 | 10:45 | Coffee (Provided) | |
| 10:45 | 12:00 | Nanopore Basics (& Data) | |
| 12:00 | 13:00 | Lunch (Provided) | |
| 13:00 | 14:00 | Clean-Up & Loading of Flow Cell | Pages 7-11 |
| 14:00 | 15:00 | Data Analysis Practical | |
| 15:00 | 15:15 | Coffee (Provided) | |
| 15:15 | 17:00 | Data Analysis Practical Continued | |
| 17:00 | 17:30 | Course Wrap-Up |
We will be fairly flexible here as we cannot always gaurentee lab work will run to time.
Nanopore sequening is a third generation sequencing technology, capable of sequencing large fragments of DNA to increasing quality.
During this course you will carry out the following:
It is our aim not to provide you with a comprehensive understanding on all nanopore sequencing has to offer, but enough of an introduction that entry into the world of long reads does not seem so daunting.
The 16S part of this course is based upon the excellent, recent, publication by Pollock et.al.: āThe Madness of Microbiome: Attempting To Find Consensus āBest Practiceā for 16S Microbiome Studies". This review provides an excellent introduction to 16S microbial species identification, and attempts to reach some conclusions about best practice.
Because the course runs over 1 day what we really want you to go away with is: * Nanopore sequencing if accessible * Nanopore sequencing is pretty straightforward * 16S sequencing is one quick application * Data analysis while somewhat challenging, is achievable.
Weāll go into more detail later, but we need to get the PCR started so that we are generating data by early this afternoon.
The practical steps today involve the following:
Letās get started with todayās experiment. Weāll talk more about nanopore sequencing and the data it produces while the PCR runs.
We will be sequencing the 16S gene of the bacterial species in your samples to help us find out what species are in the sample.
Weāll be sequencing PCR amplicons from the samples you may have brought, the community standard we have provided and also a PCR blank. So label up the correct number of 0.2ml PCR tubes.
We will be barcoding your samples. This adds a tag, unique to each sample, to the 16S amplicons. This means we can run multiple samples on the same flow cell, saving time and money. We will split the samples out bioinformatically later in the process.
You will be assigned a set of barcodes to use for your samples. We have 12 barcodes available.
This is a DNA sample from a predefined āmockā microbial community, containing 8 species of bacteria and 2 yeast. The Bacteria are present at an abundance of 12% and the yeast are present at 2%. It is provided at 10ng/μl but we have diluted this to 1ng/μl for you.
You can skip this if you have already checked using a Qubit high sensitivity assay or are just using the community standard (provided to you at 1ng/μl).
Please turn to page 6 of your handbook for the instructions. We will share a qubit flourometer and standards today.
Please turn to page 8 of your handbook for the instructions.
Remember the Community Standard is at 1ng/μl
Choose a different barcoded primer set for each sample!
Keep your reaction on ice, and weāll run them all together on the same PCR block
The PCR block already has the program installed - called ā16Sā
Once everyone has their samples ready we can select this program and hit go. We can now move on to the next part of the day.
sequencing by synthesis is responsible for >98% of the worlds sequencing data, this is the technology behind Illumina sequencing.
Two main players here:
Both of these technologies provide reads longer than Illumina, by orders of magnitude.
Pacbio (SMRT sequencing), below, still relies on the emission of light and the measurement of this signal, so the sequencer itself has a large footprint and very expensive. What is interesting about SMRT sequencing is that it reads the same molecule of DNA multiple times. In this way PacBio can obtain reasonably high accuracy. Data is not outputted in real time.
You do need a decent laptop or access to dencet commpute facilities to be able to do high accuracy basecalling.
Standard capture techniques using probes are not really suitable for long-read platforms and so both pacbio and nanopore have developed CRISPR based approaches. The nice thing with this protocol is that DNA is native and so modifications are preserved.
One really nice advantage of nanopore technology is the simultaneous collection of not only the identity of the base being sequenced but any modifications that might also be present. Having the sequence and the modfication present from the same read can be very powerful.
For DNA methylation this is a relativley straightforward analysis process:
New applications are being developed continuously, usually these are not advances in the sequencing technology, but advances in the bioinformatics analysis. Itās mostly a machine learning problem. Recently it has been shown that it is possible to detect modifications of RNA.
Here the authors have trained machine learning algorithms on synthetic modified and unmodified RNA.
āAccurate detection of m6A RNA modifications in native RNA sequencesā https://www.nature.com/articles/s41467-019-11713-9
Great starter device, cheap, portable, still get loads of data - our best run = 13Gb - 7 Million reads.
The GridION is basically an array of 5 MinIONs but with powerful compute built in.
2048 pores, available in R9.4 or the newer pore which is more accurate (R10). There are also flow cells which can sequence both strands.
Flow cells can be washed and used again. Weāve done this and been able to get just as much data on a second run. Using different barcodes is a way to ensure data is not mixed up.
Flow cells can also be ārevivedā with a nuclease flush.
āFlongle is designed to be the quickest, most accessible and cost-efficient sequencing system for smaller tests and experiments.ā 126 channels rather than the MinION flow cells 512. (504 pores)
Ā£72.50 per flow cell.